Giardia vaccine

ABSTRACT

The invention provides vaccines and methods for preventing or treating intestinal protozoal infections in an animal. In particular, vaccines and methods for prevention or treatment of giardiasis are provided. The invention also encompasses methods of preparing and methods of use of novel toxins, antibodies, vaccine strains and compositions that result from or are used in these methods.

This application is a continuation, of application Ser. No. 07/985,489,filed Dec. 4, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention relates to vaccines against intestinal protozoa.In particular, vaccines against Giardia are disclosed.

REFERENCES

The following references are cited in this application as superscriptnumbers at the relevant portions of the application.

1. Taylor, et al., Human immune response to Giardia lamblia infection,J. Infect. Dis., 155: 137-140 (1987).

2. Gillon, Changes in the small intestinal mucosa in giardiasis,"Giardia and Giardiasis", Erlandsen, et al., Eds., Plenum Press, NewYork, 163-183 (1984).

3. Wolfe, Clinical symptoms and diagnosis by traditional methods, "HumanParasitic Diseases, Volume 3--Giardiasis", Meyer Ed., Elsevier SciencePublishers, New York, 175-186 (1990).

4. Feely, et al., The biology of Giardia, "Human Parasitic Diseases,Volume 3--Giardiasis", Meyer Ed., Elsevier Science Publishers, New York,11-49 (1990).

5. Steketee, et al., Recurrent outbreaks of giardiasis in a child daycare center, Wis. Am. J. Pub. Health 79: 485-490 (1989).

6. Faubert, Evidence that giardiasis is a zoonosis, Parasitology Today,4 (3): 6614 71 (1988).

7. Roach, et al., Transmission of Giardia duodenalis from human andanimal sources in wild mice, "Advances in Giardia Research", Wallis, etal., Eds., Univ. Calgary Press, Calgary, 79-82 (1988).

8. Swabby, et al., Infection of mongolian gerbils (Merionesunguiculatus) with Giardia from human and animal sources, "Advances inGiardia Research", Wallis, et al., Eds., Univ. Calgary Press, Calgary,75-77 (1988).

9. Jakubowski, Purple burps and the filtration of drinking watersupplies, Am. J. Pub. Health, 78: 123-125 (1988).

10. Buret, et al., Zoonotic potential of giardiasis in domesticruminants, J. Infect. Dis., 155: 137-140 (1987).

11. Vinayak, et al., Systemic oral immunization with 56 kDa molecule ofGiardia lamblia affords protection in experimental mice, Vaccine 10:21-27 (1992).

12. Buret, et al., Effects of murine giardiasis on growth, intestinalmorphology and disaccharidase activity, J. Parasitol. 76 (3): 403-409(1990).

13. Buret, et al., Growth, activities of enzymes in the small intestine,and ultrasonic of microvillus border in gerbils infected with Giardiaduodenalis, Parasitol. Res. 77: 109-114 (1991).

14. Janoff, et al., The role of immunity in Giardia infections, "HumanParasitic Diseases, Volume 3--Giardiasis", Meyer Ed., Elsevier SciencePublishers, New York, 215-235 (1990).

15. Lewis, et al., Cortisone-induced recrudescence of Giardia lambliainfections in gerbils, Am. J. Trop. Med. Hyg. 36 (1): 33-40 (1987).

16. Istre, et al., Waterborne giardiasis at a mountain resort: evidencefor acquired immunity, Am. J. Public Health, 74 (6): 602-604 (1984).

17. den Hollander, et al. Immunology of Giardiasis. Parasitology Today.4: 124-130 (1988).

18. Adam. The biology of Giardia spp. Microbiology Reviews. 55: 706-732(1991).

19. Roberts-Thomson, et al. Acquired resistance to infection in ananimal model of giardiasis. J. Immunology. 117: 2036-2037 (1976).

20. LoGalbo, et al. Symptomatic giardiasis in three patients withX-linked agammaglobulinemia. J. Pediat. 101: 78-80 (1982).

21. Janoff, et al. Acute antibody responses to Giardia lamblia aredepressed in patients with AIDS. J. Infect. Dis. 157: 798-804 (1988).

22. Butscher, et al. The therapeutic action of monoclonal antibodiesagainst surface glycoprotein of Giardia muris. Immunology. 64: 175-180(1988).

23. Stevens, et al. Local immunity in murine giardiasis: is milkprotective at the expense of the maternal gut? Trans. Assoc. Am. Phys.91: 268-272 (1978).

24. Roberts-Thomson, et al. Protection of mice against Giardia murisinfection. Infect. Immun. 24: 971-973 (1979).

25. Smith, et al. Chronic giardiasis: studies on the drug sensitivity,toxin production, and host immune response. Gastroenterology. 83:797-803 (1982).

26. Katelaris, et al. Diarrhea and malabsorption in giardiasis: amultifactorial process? Gut. 33: 295-297 (1992).

27. Nash, et al. Excretory-secretory products of Giardia lamblia. J.Immunology. 131: 2004-2010 (1983).

28. Katelaris, et al. Pathogenesis of diarrhea caused by Giardialamblia: evidence for an exotoxin. J. Gastroenterol. Hepatol. 3(suppl1): A4 (1988).

29. Zaman, Dextran particles as a carrier for Giardia lamblia forscanning electron microscopy, J. Electron. Microsc. 41: 179-180 (1992).

30. Kirkpatrick, et al., Feline giardiasis, observations on natural andinduced infections, Am. J. Vet. Res., 45: 2182-2188 (1984).

The disclosure of the above publications and patents are hereinincorporated by reference in their entirety to the same extent as if thelanguage of each individual publication or patent were specifically andindividually included herein.

BACKGROUND OF THE INVENTION

Intestinal protozoa are the cause of many human and animal diseases.When they infect domestic animals, severe economic losses may result.Intestinal protozoa of importance include cryptosporidium, trichomonads,histomonas, spironucleus, entamoeba, coccidia, toxoplasma andsarcocystis. One of the most problematic intestinal protozoa is Giardia.

Giardia lamblia is the most commonly found pathogenic parasite inwestern countries and is endemic in much of North America¹. Giardia is aflagellated protozoan which is transmitted through the fecal-oralroute². The mechanism of pathogenesis in Giardia is poorly understoodbut it results in symptoms similar to many other gastrointestinalailments. The most common symptoms are diarrhea, anorexia, malaise,abdominal distention and flatulence. Acute stages of the infectionusually last only a few days although occasionally, especially inchildren, the chronic stage may last for months³.

Once inside its host, the Giardia trophozoites attach to the epitheliumof host intestinal villi where they multiply, become encysted, and thenare shed in the host feces. Some infected hosts become asymptomatic cystpassers after the short acute stage of the infection has passed⁴.

A major source of Giardia infection is contaminated drinking water. Thishas earned it the name "backpackers diarrhea" because of the largenumber of people infected due to exposure to contaminated water whencamping⁴. Outbreaks of giardiasis are also a frequent problem in daycare centers. Recurring Giardia infections in day care centers arecommon, and many of the children are found to be carriers of theinfection although they show no symptoms. This makes detection andeffective treatment of Giardia in day care centers difficult⁵.

A major area of interest in Giardia infections is the ability of animalhosts to act as reservoirs for human infective strains of Giardia. Thereis evidence that inter-species transmission of Giardia can and doesoccur. Human strains of Giardia have been shown to be infective ingerbils, mice, guinea pigs, raccoons, and beavers⁶⁻⁸. The common namegiven to Giardia infection in Canada, "beaver fever" was born when anepidemic of the infection resulted from the contamination of a watersource by a family of three beavers⁹. Giardia infections are common indogs and cats. It is estimated that 10-68% of dogs and 25% of cats areinfected with Giardia. Studies of the prevalence of Giardia infection indomestic ruminants found infection in 17.7% of sheep and 10.4% ofcattle, with the incidence being higher in lambs (35.6%) and calves(27.7%)¹⁰.

As previously stated, clinical giardiasis may range from asymptomaticcarriage of the parasite to an illness characterized by diarrhea,abdominal cramps, headache, gas, bloat, dehydration and malaise¹,11.Weight loss and retardation of growth are also common problemsassociated with giardiasis in humans and animals¹¹.

In humans, histological changes associated with Giardia include villusatrophy². More recent studies using Mongolian gerbils as an animal modelhave shown diffuse shortening of the microvillus mainly in the duodenumas well as a decrease in the brush border enzyme activity. The enzymedeficiencies may be caused by the shortening of the epithelialmicrovilli¹². Other studies have correlated Giardia infection withdecreased weight gain, decreased food intake, decreased intestinaldisaccharidase activities and villus atrophy¹³. The diffuse shorteningof microvillus may be the result of a toxin produced by the Giardia¹².

Given the prevalence of giardiasis and the difficulty in treating allthose who are infected due to asymptomatic carriers, the development ofa vaccine against Giardia is highly desirable. It has been shown thathosts will raise an immune response to Giardia and that they can retainlong lasting immunity after the primary infection¹⁴, 15. If a host isrepeatedly exposed to Giardia, the risk of infection decreases¹⁶.Therefore, it is possible for a host to acquire immunity. The immuneresponse of the host may explain the wide variability of host responsesto Giardia. Sickness results when the immune system is unable togenerate an adequate defense against the protozoan¹⁴. Previous studiesin the area of Giardia immunity have been hampered by the lack ofunderstanding of pathogenic mechanisms.

It has been demonstrated in natural and experimental Giardia lamblia andGiardia muris infections that both cellular and humoral immunity aregenerated by the host¹, 3, 14, 17, 18. In natural and experimentalinfections, development of immunity has been associated with eliminationof the parasite¹⁶, 19. However, humans and animals with an elevatedimmune response to Giardia may still have clinical or subclinicalgiardiasis which may be due to inadequate immunity in the host¹⁴, 20,21. It has been shown in humans and in animal models that severity ofsymptoms, course of infection, and infectivity rates are reducedfollowing a second exposure to Giardia parasites¹⁴, 16, 19.

There have been numerous monoclonal and polyclonal antibodies producedin laboratory animals to Giardia lamblia and Giardia muris trophozoitesand cysts. The purposes of producing these antibodies are for diagnosticpurposes or reagents for laboratory studies. These antibodies areproduced by employing standard hybridoma technology and using BALB/cmice. Polyclonal antibodies have been produced in rabbits and smallrodents by performing multiple immunizations with antigens and Freund'sadjuvant.

Passive immunization with anti-Giardia antibodies have been conducted.Butscher, et al.²² demonstrated that intraperitoneal administration ofmonoclonal antibodies to a surface glycoprotein of G. muris trophozoitesreduced parasitic burden in mice. Passive transfer of immunity has beendemonstrated in mothers' milk²³. Mother mice previously infected withGiardia muris were able to confer protection upon their sucklingoffspring while the milk of naive mothers was not protective.

There are extremely limited studies of active immunization of animalswith Giardia trophozoites or with sub-unit vaccines. Roberts-Thomson etal. conducted a study where two strains of laboratory mice werevaccinated and challenged²⁴. Intact Giardia muris trophozoites (10⁶)were combined with Freund's adjuvant (1:1 ratio) and injectedintraperitoneally and in the footpad. Four weeks later animals wereboosted in the same sites with the same dose without adjuvant. Controlmice received only adjuvant or were untreated. One week later mice werechallenged with Giardia muris cysts. Vaccinated BALB/c mice had areduced cyst output for a shorter duration while there was no differencein the cyst output between control and vaccinated C3H/He mice. Thus,this study produced variable and ineffective results.

Vinayak et al.¹¹ isolated a 56 kDa protein from Giardia lamblia. Micewere subcutaneously immunized (Day 0) and orally immunized (day 7) with100 μg of multilammelar phosphatidylcholine liposomes (MPL)-entrapped 56kDa surface associated antigen. Unimmunized and animals similarlyimmunized with MPL-entrapped PBS (Phosphate buffered saline) served ascontrols. All animals were challenged seven days after the lastimmunization dose. Immunization with MPL-entrapped 56 kDasurface-associated antigen resulted in a reduction in trophozoitecolonization of the gut and duration of infection when compared to thecontrol groups. It was suggested that the 56 kDa surface antigenimmunoregulates the Giardia infection.

It is believed that Giardia secretes cytotoxins that influence thefunction or structure of the small intestinal mucosa²⁵, 26, but therehas yet to be one or more toxic principles identified using classicalmethods for identification of cytotoxins²⁵, 26. Culture filtrates ofGiardia have been shown to elaborate excretory/secretory products intothe culture medium²⁵. Culture filtrates have been shown to damagefibroblasts in culture as well as reduce salt and water absorption fromperfused loops of rats but the role of these substances in thepathogenesis of giardiasis is unknown²⁷.

There exists a need for an effective vaccine which can be used toprevent and treat protozoal infections, including giardiasis, inanimals, including humans.

SUMMARY OF THE INVENTION

The invention provides vaccines and methods for preventing or treatingintestinal protozoal infection in an animal, as well as novel toxinswhich may be used in said vaccines. Vaccines and methods for preventingor treating giardiasis, in particular, are also provided. The inventionalso encompasses methods of preparing and methods of use of noveltoxins, antibodies, vaccine strains and compositions that result from orare used in these methods.

Accordingly, in one aspect, the invention provides vaccine strains ofintestinal protozoa. A method of preparing these vaccine strains is alsoprovided.

Another aspect of the invention is a vaccine composition comprising avaccine strain of an intestinal protozoan. A cell free vaccinecomposition comprising a subunit or toxin of an intestinal protozoan isalso provided, as is a method of preparing these vaccine compositions.

A further aspect of the invention is a method of preventing or treatinginfection by intestinal protozoa in an animal comprising administeringto the animal an effective amount of a vaccine strain of the intestinalprotozoa. A method of preventing or treating infection by an intestinalprotozoan comprising administering a cell free composition comprising asubunit or toxin of the intestinal protozoan is also provided.

Yet another aspect of the invention is a toxin of an intestinalprotozoan. A method of preparing the toxin is also provided, as are anantibody to the toxin and a method of passive immunization using theantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. illustrates the growth of various strains of Giardia.

FIG. 2. illustrates the toxin titer of Strain S2 of Giardia in CHOcells.

FIG. 3. illustrates the toxin titer of Strain WB of Giardia in CHOcells.

FIG. 4. illustrates the toxin titer of Strain D3 of Giardia in CHOcells.

FIG. 5. illustrates the effect of vaccination on weight gain in kittensfollowing challenge with Giardia.

FIG. 6. illustrates the effect of vaccination on immune response inkittens.

FIG. 7. illustrates the effect of vaccination on trophozoite count inkittens following challenge with Giardia.

FIG. 8. illustrates the effect of vaccination on the percentage ofkitten gut samples in which trophozoites are seen following challengewith Giardia.

FIG. 9. illustrates the excretion of fecal cysts in vaccinated andunvaccinated dogs.

FIG. 10. illustrates the effect of Giardia toxin on CHO cells.

FIG. 11. illustrates a toxin production curve.

FIG. 12. illustrates the effect of bile on the production of Giardiatoxin as measured by CHO cell elongation.

DESCRIPTION OF THE INVENTION

A. Definitions

As used herein, the following terms have the following meanings:

Adjuvant: a vehicle used to enhance antigenicity. The use of adjuvantsis well-known in the art. Adjuvants may include suspensions of mineralson which antigen may be adsorbed, such as alum, aluminum hydroxide orphosphate; water-in-oil emulsions in which antigen solution isemulsified in mineral oil, such as Freund's incomplete adjuvant; and mayinclude additional factors, such as killed mycobacteria in Freund'scomplete adjuvant, to further enhance antigenicity.

Antibody: a molecule, especially a protein, that binds immunologicallyto a known antigen or a determinant of an antigen.

Bile: the substance secreted by the liver and discharged into theduodenum where it aids in the emulsification of fats, increasesperistalsis, and retards putrefaction. The term bile is also used torefer to powdered or dried bile. Bile also means any component of bile,such as an individual bile salt.

Colonization: attachment to the gut of the infected animal by theprotozoan.

Cyst: the infectious form of many protozoal parasites, such as Giardia.Cysts are usually provided with a highly condensed cytoplasm andresistant cell wall. They are often shed in the feces, and this is theway in which the disease is spread from one animal to another. Cysts maybe viable, i.e. able to produce a trophozoite in a new host, or may benon-viable.

Effective Amount: dose required to protect an animal against infectionor disease or alleviate a particular symptom of an infection or disease.

Feed Conversion: measure of an animal's ability to gain weight expressedas the weight of feed required to produce a unit quantity of bodyweight. Intestinal diseases, including those caused by protozoa, reducethis parameter by making an animal less efficient in converting feed tobody weight.

Giardia: a genus of parasitic flagellates that parasitize the smallintestine. As used in this application, the term includes all species ofthis genus. The genus Lamblia, formerly used to refer to Giardia, isalso included in this term as used in this application.

Giardiasis: infection with Giardia. The symptoms of the infection, suchas diarrhea, abdominal cramps, headache, gas, bloat, weight loss, lackof weight gain, dehydration, malaise, malabsorption, colonizing of thegut with the parasite, shedding of cysts, etc. are also included in theterm giardiasis.

Immune Response: development in the host of a cellular and/orantibody-mediated immune response to a composition or vaccine ofinterest. Such a response may consist of one or more of the following:producing antibodies, B cells, helper T cells, suppressor T cells,and/or cytotoxic T cells directed specifically to an antigen or antigensincluded in the composition or vaccine of interest.

Intestinal Protozoa: any protozoa which inhabits the gut of the animalit infects.

Intestinal Protozoal Infection: infection with an intestinal protozoa.The symptoms of the infection, such as diarrhea, abdominal cramps,headache, gas, bloat, weight loss, lack of weight gain, dehydration,malaise, malabsorption, colonizing of the gut with the parasite,shedding of cysts, etc. are also included.

Neutralize: able to prevent or alleviate toxic effects.

Prevention of symptoms: includes prevention of any effect caused by atoxin.

Production in vitro: production in culture, not in an infected hostanimal. Production in vitro includes recombinant production.

Protectively Immunogenic: able to protect an animal against infection ordisease or alleviate a particular symptom of an infection or disease.

Recombinantly produced: produced by means of gene expression in anyother system including microorganisms, plants or animals and/orchemically synthesized by methods known in the art when the sequence isknown.

Sonication: disruption of cells by exposing a suspension of the cells tohigh frequency sound waves.

Subunit: any part of an intestinal protozoa which is less than the wholeorganism. Subunits that are antigenic may be used in vaccinecompositions to produce an immune response. Subunits may includeflagella, the ventral adhesive disk, membranes, cytoskeletal membraneproteins, cytosolic membrane proteins, toxins and any other part of anorganism which may be antigenic and induce an immune response. Thisincludes recombinantly produced subunits.

Toxin: a noxious or poisonous substance that is produced by anintestinal protozoa. It may be an extracellular product (exotoxin). Whenintestinal cells are affected, a toxin is classified as an enterotoxin.This includes recombinantly produced toxins.

Trophozoite: the vegetative form of certain intestinal parasites, suchas Giardia.

Vaccine Strain: a strain of an intestinal protozoa which is protectivelyimmunogenic when administered to an animal. This includes strains whichhave been genetically attenuated.

B. Detailed Description of The Invention

This invention provides vaccine strains of intestinal protozoa andtoxins from said protozoa. It has unexpectedly been found that culturingintestinal protozoa in media containing bile makes them protectivelyimmunogenic when used to vaccinate animals. Further, said culture inbile-containing media provides for production, in vitro, of a toxin.This toxin may be used for immunization against infection by theintestinal protozoa or for raising toxin-neutralizing antibodies.

In particular, intestinal protozoa which are present extracellularly inthe gut of the host animal are preferred for use in the presentinvention. In a most preferred embodiment, the invention providesvaccines strains, toxins, and antibodies to toxins of Giardia. Suchstrains, toxins and antibodies are useful for preventing and treatinggiardiasis and the symptoms thereof.

A variety of Giardia strains are useful in the present invention.Strains which grow well in vitro, are able to infect target animalspecies, and produce a toxin when grown in vitro are preferred. Inparticular, Giardia strains WB (a human isolate), S2 (a strain which weisolated from sheep), D3 (a strain which we isolated from dogs), and N(a strain which we isolated from drinking water) may be used. Strains S2and D3 are most preferred.

The Giardia strains of this invention were cultured by growing them inTYI-S-33 media containing bile. Dehydrated bovine bile was generallyused, however it is anticipated that fractions of bile, includingindividual bile salts, will be useful in the present invention. TheGiardia trophozoites may be grown in containers which also containfinely divided solid supports. This increases the surface area to whichthe trophozoites may attach. Dextran beads are a particularly preferredsolid support²⁹. Other support systems such as glass beads or fibers mayalso be suitable. Details of media preparation, Giardia trophozoitesubculture and harvest are set forth in the Examples below.

The invention also provides vaccine compositions comprising a vaccinestrain of Giardia or other intestinal protozoa which is effectivelyimmunogenic. Various strains of Giardia may be useful in such vaccinecompositions. In particular, strains which produce a toxin when culturedin vitro are preferred. Examples of most preferred strains are Giardiastrains S2 and D3.

The vaccine strains may be cultured as set forth in the Examples below,then harvested for use in vaccine compositions. Protozoa may bedisrupted before use in vaccine compositions. Various methods ofdisruption may preferably be used, including sonication, osmosis, theuse of pressure differentials, or freezing. Sonication is mostpreferred.

Vaccine compositions may contain one or more vaccine strains of anintestinal protozoan and/or one or more subunits and/or toxins ofGiardia or other intestinal protozoa. Such subunits and/or toxins may beused in addition to whole or sonicated protozoa or may be used incell-free vaccine compositions.

It may be useful to inactivate the intestinal protozoa, toxins orsubunits before use in vaccine compositions. Conventional techniquessuch as mild heat treatment or formalin inactivation may be used.

The formulation of such vaccine compositions may include suitablepharmaceutical carriers, including adjuvants. The use of an adjuvant,such as an alum-based adjuvant, is preferred. Many commercial adjuvantswould be useful in the present invention. For these studies, analum-based adjuvant containing aluminum hydroxide and Quill A (SuperFos, Copenhagen, Denmark), was used. Exact formulation of said vaccinecompositions will depend on the particular vaccine strain, the speciesto be immunized and the route of immunization. Such vaccine compositionformulation is well-known to those skilled in the art.

Such vaccine compositions are useful for immunizing any animalsusceptible to intestinal protozoa, such as bovine, ovine, caprine,equine, leporine, porcine, canine, feline and avian species. Bothdomestic and wild animals may be immunized, and immunization of foodproducing animals is contemplated. Humans, may also be immunized withthese vaccine compositions.

The present invention provides a method of preventing or treatinggiardiasis or other intestinal protozoal infection by administering aneffective amount of a vaccine strain of Giardia or other intestinalprotozoa to an animal in need of such prevention or treatment. Suchvaccine strain may be used in a vaccine composition as previouslydiscussed. This method is useful in dogs, cats, humans, domestic animals(especially food producing animals), avian species, and wild animals.Use in wild animals may prevent contamination of water supplies used byhumans or domestic animals.

The route of administration may be any convenient route, and may varydepending on the intestinal protozoan, the animal to be treated, andother factors. Parenteral administration, such as subcutaneous,intramuscular, or intravenous administration, is preferred. Subcutaneousadministration is most preferred for canine and feline species. Oraladministration may also be used, including oral dosage forms which areenteric coated.

The schedule of administration may vary depending on the intestinalprotozoa and the animal to be treated. Animals may receive a singledose, or may receive a booster dose or doses. Annual boosters may beused for continued protection. In particular, two doses 21 days apartare preferred as a primary course.

The age of the animal to be treated may also affect the route andschedule of administration. Administration is preferred at the age whenmaternal antibodies are no longer present and the animal isimmunologically competent. This is about 6 to 7 weeks of age in canineor feline species. Additionally, immunization of mothers to preventinfection of their offspring through passive transfer of antibodies intheir milk is preferred.

The method of this invention is effective in preventing colonization ofthe gut, i.e. preventing attachment of the protozoa to the gut mucosa.It is also effective in preventing symptoms of giardiasis. This includesneutralizations of toxin and prevention of any physiological toxineffects which may occur when the organisms may be present in the lumenof the gut, but not attached. Further, the method of this inventiondecreases fecal shedding of cysts, and in particular, viable cysts. Thisprevents further spread of infection.

Treatment may be administered to symptomatic or asymptomatic animals,including animals or humans with chronic infection, and may be used toincrease growth rate by alleviating such symptoms of infection asdiarrhea. Thus, when administered to a food producing animal, it mayincrease feed conversion.

The present invention provides toxins of Giardia or other intestinalprotozoa and methods of producing these toxins. It has unexpectedly beenfound that culturing organisms in media containing bile causes them toproduce a toxin in vitro. This toxin may be used to immunize animalsagainst intestinal protozoal infection. When the media in which theseorganisms were grown was concentrated, a toxin useful for preventingand/or treating infection was obtained. This toxin in Giardia is anexotoxin and highly cytotoxic. It appears to be a potent enterotoxin.

Until recently, attempts to isolate a Giardia enterotoxin had beenunsuccessful. But the use of concentrated solutions and a more sensitiveassay system has allowed the detection of a cytotoxic factor that isproduced by Giardia. The toxin was detected using Chinese Hamster Ovary(CHO) cells. The CHO bioassay has been determined to be 100-10,000 timesmore sensitive than skin permeability, fat cell lipolysis and ileal loopassays. Tests with the toxin have shown it to cause CHO cell elongation.The amount of elongation (i.e. the percentage of cells elongated) wasfound to generally increase with toxin concentration. The resultsobserved with the Giardia toxin are very similar to other work done withbacterial enterotoxins. A similar effect on CHO cells has been foundusing E. coli toxins, cholera toxin, and pertussis toxin.

Partial purification of the Giardia toxin has been achieved. It has beenestablished that the Giardia toxin is an intracellularly producedprotein which is stable at 37° C., at room temperature, whenrefrigerated, or when frozen. Using SDS-PAGE, it has been establishedthat media containing toxin contains Coomassie-staining proteins ofapproximate molecular weight of 22, 32, 38 and 39 kDa, as well asseveral proteins of molecular weight greater than 97 kDa, which are notpresent in control media.

Immunization with the toxin reduced symptoms of giardiasis, includingdiarrhea and decreased fecal cyst shedding.

The present invention also provides antibodies to toxins of Giardia orother intestinal protozoa. Polyclonal antibodies have been raised toGiardia toxin. Monoclonal antibodies may also be raised by conventionaltechniques. These antibodies will be useful as an antiserum toneutralize the toxic effects of the toxin. Thus, they may be expected torelieve symptoms of giardiasis or other intestinal protozoal infection.It is expected that oral administration of these antibodies using anenteric coated dosage formulation will be preferred.

The methods of this invention will be useful to produce vaccine strains,vaccine compositions, toxins, and anti-toxins for preventing andtreating other intestinal protozoal infections. Other intestinalprotozoa useful in the present invention include cryptosporidium,trichomonads, histomonas, spironucleus, entamoeba, coccidia, toxoplasmaand sarcocystis. In particular, cryptosporidium, trichomonads,histomonas, spironucleus and entamoeba are preferred.

Cryptosporidium muris (also called Cryptosporidium parvum) is aprotozoan parasite found in the small and large intestine of mammals andpoultry and Cryptosporidium melegridis is found in the brush border ofthe respiratory and gastrointestinal tract of poultry. The parasitecauses mild to severe diarrhea in humans, other mammals and birds, andrespiratory disease in birds. Cryptosporidium causes a reduction inbrush border surface area, impaired electrolyte transport and amalabsorptive diarrhea similar to giardiasis. There is a tremendouspotential for the development of a whole cell or subunit vaccine forCryptosporidium spp. This vaccine can be prepared by disrupting theparasite, combining these proteins with adjuvant and immunizing animalsor humans by an oral or a parenteral route. One or more toxins may be animportant virulence factor and these toxins (or toxoids) can also beused to vaccinate by oral or parenteral routes. The vaccine may be usedto prevent the disease or protect against the symptoms of the disease.Anti-toxin antibodies may be used to treat the symptoms ofcryptosporidiosis.

Trichomonads are a large family of flagellated protozoan parasites whichare found in the gastrointestinal, reproductive and respiratory tractsof mammals and birds. Most trichomonads are believed to benon-pathogenic commensals. However, we believe that like Giardia, someof these protozoan parasites may influence weight gain and feedconversion in animals and in some cases may cause diarrhea. Themechanism may be toxin mediated. Vaccination with disrupted trichomonasparasites and adjuvant may provide protection from infections or reducesymptoms of the infection. Similarly a toxin or toxoid may provideprotection. The vaccine would prevent colonization of the gut and theinjury to the gut. There may be cross protection of differenttrichomonad species.

Histomonas meleagridis is a flagellated protozoan parasite of poultry.The parasite is released from the cecum and passes by way of the bloodstream to the liver. There is necrosis and tissue degeneration of theliver and cecum. Birds that have recovered from the infection are immuneto the disease. We believe a toxin may be involved in the pathogenesisof the disease. Vaccination with a disrupted cell or toxin vaccine mayprovide an effective method of control of histomoniasis in poultry. Thevaccine would prevent colonization of the gut or penetration of thececum. The vaccine may also prevent the necrosis of the liver and cecum.

Spironucleus meleagridis is a disease of young birds resulting indiarrhea and death. The gut is found filled with a watery fluid on postmortem. This parasite infects domestic and wild birds. We believe thatSpironucleus meleagridis induces symptoms through a toxin mediatedprocess. A disrupted whole parasite or toxin or toxoid vaccine mayprovide protection from this infection. The vaccine would neutralize thetoxin and/or prevent colonization of the parasite.

Entamoeba histolytica is a pathogenic amoebic protozoan which causesamoebic dysentery in humans and animals. The organism colonizes thececum and colon causing diarrhea and dysentery. The parasite may invadethe gut wall and multiply in the submucosa causing ulceration. Theparasite may also enter the lymph ducts or mesenteric veins resulting inabscesses throughout the body. We believe that one or more toxins may beimportant virulence factors. Vaccination using disrupted trophozoitesand/or toxin may act to provide protection against colonization and/orinvasion of the parasite. Vaccination may also protect against thediarrhea and dysentery seen in this disease, as it would neutralize thetoxin and/or prevent invasion of the parasite.

Coccidiosis is a complex intestinal disease induced by Eimeria spp. orIsospora spp. and is of major economic importance in domestic animals.These protozoans have a complex life cycle and are host specific.Attempts to produce vaccines have been made. Poultry vaccines have beenproduced (CocciVac, CocciVac T, Sherwin Laboratories). These vaccinesinclude all the pathogenic species for chicken and turkeys. Developmentof subunit vaccines have been largely unsuccessful. A coccidia toxin hasnot been identified and toxin vaccines have not been produced. There isa potential to produce a subunit or fortified vaccine based on a toxinor toxoid for coccidia of humans and animals. These include: Isosporaspp., Eimeria spp., Wenyonella spp. and Tyzzeria spp.

Toxoplasma have a complex life cycle. Several investigative groups havebeen unable to protect mice, hamsters and rabbits by experimentalimmunization with heat-killed and heat- or formalin- killed homologoustachyzoites, with or without adjuvant. Certain fractions have providedprotection against experimental challenge. A cytotoxin of Toxoplasma hasnot been identified; however, immunization with a toxin or toxoid mayprovide protection from this disease in animals and humans.

Like coccidia, Sarcocystis have a complex life cycle with the oocystsbeing present in the predators' intestinal cells and during the asexualstage in the tissues of the prey animal. They are common in manydomestic and wild animals. At the present time there is no vaccine forthese protozoan parasites and a cytotoxin has not been demonstrated.There is potential for vaccine development for this parasite using thepresent invention.

The following examples are not intended to limit the scope of theinvention in any manner.

C. Examples of Embodiments of The Invention

In general, the following materials and methods were used in theseexamples unless otherwise noted:

1. Media recipe and preparation

Media for G. lamblia (TYI-S-33) was prepared using the followingingredients.

    ______________________________________                                                              g/l                                                     ______________________________________                                        Casein Hydrolysate (Gibco 152-0014M)                                                                  20.0                                                  Yeast Extract (BBL 11929)                                                                             10.0                                                  Dextrose (anhyd.)       10.0                                                  NaCl                    2.0                                                   K.sub.2 HPO.sub.4 (anhyd.)                                                                            1.0                                                   KH.sub.2 PO.sub.4 (anhyd.)                                                                            0.6                                                   L-Cysteine (Sigma C 2529)                                                                             2.0                                                   L-Ascorbic Acid (Sigma A 4034)                                                                        0.2                                                   Bovine Bile (Sigma B 3883)                                                                            0.8                                                   ______________________________________                                    

Chemicals were from British Drug Houses (BDH) unless otherwisespecified.

The above dry ingredients were combined and stored in the dark at 4° C.until needed. National Collection of Type Cultures, Colindale, England,(NCTC) vitamin mix (Gibco Cat. #440-1100 EB) was prepared according topackage instructions, sterile filtered (0.22 μ) and stored as 30 mlaliquots at -20° C. A solution of ferric ammonium citrate was preparedby suspending 2.28 g in 50 ml distilled water (dH₂ O) using a volumetricflask. The solution was stored in the dark at 4° C., as it isphotolabile. CLEX (Fetal Calf Serum (FCS) substitute) was thawed, thenstored at -20° C. as sterile 100 ml aliquots (Dextran Products CLEXC-500).

For one liter of media, half of 870 ml dH₂ O was poured into a 1L flaskwith stirring bar, dry ingredients were added, and the remaining waterused to rinse the container and flask edges. Then 500 ml of the ferricammonium citrate solution was added. The solution was stirred until themedia was transparent (usually 2-3 hours), then the pH adjusted to 6.8with 5M NaOH. The media was sterile filtered (0.22 μ and prefilter)under aseptic conditions, 30 ml of vitamin mix and 100 ml of CLEX addedand mixed. Prepared media was aliquoted and stored at -20° C. untilneeded.

2. Freezing Giardia trophozoites

Giardia trophozoites were frozen using the following procedures. Giardiawere grown into late log phase (72 hours). Media was poured out so as toretain the healthy trophozoites which were on the sides of the tube.Fresh media was poured in, and the tubes cold shocked on ice for 10-15minutes, then centrifuged at 500× g for 10 minutes at 4° C. Understerile conditions, 14 ml of media was pipetted off, leavingapproximately 2 ml for resuspension of the trophozoites. About 0.9 ml ofthis trophozoite suspension was added to cryotubes containing 0.9 ml of20% DMSO in CLEX. The cryotubes were placed into canes for storage inliquid N₂ and placed in an insulated container. The insulated containerwas placed into a -70° C. freezer for a minimum of 12 hours and amaximum of 72 hours, then into the liquid N₂ freezer.

Trophozoites were quick thawed by placing cryotubes into a 37° C. waterbath, placed into fresh media and incubated horizontally for the firstday, subcultured after 24 hours and incubated as usual.

3. Subculturing Giardia trophozoites

The Giardia lamblia strains used were: WB (a human isolate, ATCC 30957),S2 (a strain isolated from sheep in our laboratory, D3 (a strainisolated from dogs in our laboratory), and N (a strain isolated fromdrinking water from Botwood, Newfoundland in our laboratory).

The procedures following were used to subculture Giardia trophozoites ofall strains used. Giardia were grown into late log phase (72 hrs), thencold shocked by placing culture tubes into ice for 10 to 15 minutes.Under a laminar flow hood, approximately 7-8 mg of Piperacillin(Pipracil, Lederle) was added to each new culture tube, then 15 ml offresh TYI-S-33 media was added to each new tube. After cold shocking,tubes with late log Giardia were inverted a few times to mix up thesettled and adherent populations. With a sterile 1 ml pipette, 1 ml oftrophozoites was removed and added to the new culture tube, flushing thepipette 1 to 2 times. New tubes were sealed with Parafilm and placedupright in a 37° C. incubator. Subculture was done again in 3 to 4 days.

Alternatively, Giardia trophozoites were cultured in double surfaceglass roller bottles (Bellco Cat. #7730-38910). Concentrations of 10⁶trophozoites per ml culture medium are easily obtained after 72 hours ofincubation. Each roller bottle required approximately 650 ml of media.In order that the bottle be full enough to extrude any large airbubbles, the media was warmed to 37° C. before use. To avoid exposingtrophozoites to a high concentration of Piperacillin at the top of thebottle, the antibiotic was first dissolved in some media (toapproximately 0.5 mg/ml final concentration), the solution poured intothe bottle, the bottle filled to near the top with more media, Giardiaadded (a minimum of 10⁶ trophozoites, cold shocked as above), the bottlefilled to the top, any air bubbles removed using a pipette, and thebottle tightly sealed with Parafilm. The roller bottle apparatus(Wheaton Model III) was set to 6-8% of motor output (two revolutions perminute) and bottles cultured for 3-4 days.

4. Harvesting Giardia trophozoites

Giardia trophozoites were harvested as follows. Giardia were grown intolate log phase (72 hrs), then cold shocked by placing tubes or bottleson ice (15-20 minutes for 10 and 30 ml tubes, 45 minutes for rollerbottles). Tubes or bottles were then inverted several times, and thecontents poured into sterile centrifuge tubes or bottles. These werespun at 500× g, at 4° C. for 10 minutes, to pellet the trophozoites.Media was removed and the pellet resuspended in sterile PBS (pH 7.2) tothe original volume of the media. The suspension was spun at 500× g, 4°C., for 10 minutes. Supernatant was removed and the trophozoitesresuspended in fresh PBS. A total of four PBS washes was performed.After the final spin, the trophozoites were resuspended to the desiredconcentration in PBS.

The trophozoites were centrifuged at low speed so as not to damage them,thus the pellet formed is very soft and the organisms are motile. It isimportant to move quickly to remove the supernatant before the pelletresuspends itself.

5. Sonicating Giardia trophozoites

Giardia trophozoites were sonicated using a Virsonic Cell Disrupter.Three 20 second bursts were generally sufficient. The presence of intacttrophozoites was checked using a hemocytometer. An additional burst wasused where necessary for complete disruption. Trophozoites were kept onice at all times, and the sonicator tip cooled with 70% ethanol betweenbursts. Sonicates were aliquoted and stored at -20° C.

6. Preparing whole sonicate vaccine

Whole sonicate vaccine of Giardia was prepared as follows. The proteinconcentration of the sonicate was determined (BIORAD Protein Assay) andadjusted to 0.75 mg/ml in sterile PBS. This solution was mixed 4:1 withthe previously described alum-based adjuvant for use in immunizinganimals in the following studies.

7. Concentrating Giardia toxin.

Giardia medium and very dilute samples were concentrated to achieve auseful concentration of toxin using an Amicon apparatus (Model No.8200). This device uses N₂ pressure to force water and low molecularweight (MW) particles through a membrane with a specified MW cutoff. Themembrane YM-10, used for these studies, has a MW cutoff of 10,000. Theapparatus also contains a stir bar which helps to keep proteins fromplugging the membrane, allowing water to pass through more easily.

Membranes were sometimes used more than once for the same protein. Afteruse, the membrane was rinsed in dH₂ O and refrigerated in 10% ethanol.New membranes were soaked in dH2O for 1 hour with three water changes toremove the membrane preservative.

8. Young kitten model

An investigation into the feasibility of using young (6-8 week) kittensas a model infection system was performed as our initial study. Multiplefecal flotations were performed on feces of these kittens during theirconditioning period to ensure giardia-free status. Kittens (n=6) wereanesthetized, laparotomies were performed and the kittens wereinoculated intraduodenally with 1.0×10⁶ Giardia lamblia trophozoites in1.0 ml PBS. Each of two kittens received the following Giardia lambliastrains: 1) WB strain (a well characterized human isolate, ATCC 30957);2) S2 strain (a strain isolated from a sheep by our laboratory); or 3)D3 strain (a strain isolated from a dog by our laboratory).

Clinical signs were monitored and four fecal examinations for cystsperformed during the 7 days immediately following infection (see Table1). Serum samples were taken on the day of experimental infection andthe 7th day (the day of post mortem) for determination of IgM titer andIgG titer (Table 2) to the respective sonicated Giardia strains (i.e.WB, S2, D3 respectively). Trophozoite counts were performed on 1.0 cmlengths of duodenum from each kitten (Table 3) and tissue samples takenfor light microscopy and electron microscopy. Bile and mucosal scrapingswere collected and stored frozen (-70° C.) for future localimmunological response testing and enzymatic assays.

Protozoan parasites with morphology typical of Giardia lamblia were seenin the duodenum, jejunum and (to a lesser extent) ileum of WB and S2infected kittens. No trophozoites were evident in D3 infected kittensusing any of the microscopy techniques. Based on this pilotinvestigation, it was decided to use the kitten infection model forfurther studies, since Giardia strains are infective and produceclinical signs in this model.

                  TABLE 1                                                         ______________________________________                                        Clinical Signs of Giardia Infected Kittens                                    KITTEN  Giardia   SYMPTOM     FECAL RESULT                                    NUMBER  STRAIN    PRESENT     FOR GIARDIA                                     ______________________________________                                        1       WB        SOFT STOOL  NEGATIVE                                        2       WB        SOFT STOOL  NEGATIVE                                        3       S2        DIARRHEA    POSITIVE                                        4       S2        SOFT STOOL  POSITIVE                                        5       D3        DIARRHEA    NEGATIVE                                        6       D3        SOFT STOOL  NEGATIVE                                        ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Kitten Serum ELISA Titers to Giardia                                                                   DAY 7                                                              Giardia  DAY 0 TITER TITER                                      KITTEN NUMBER STRAIN   IgM     IgG   IgM  IgG                                 ______________________________________                                        1             WB       8       1     8    256                                 2             WB       16      0     16   16                                  3             S2       64      4     64   128                                 4             S2       64      0     64   64                                  5             D3       64      2     32   640                                 6             D3       32      4     32   256                                 ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Duodenal Tissue Trophozoite Counts                                                         Giardia                                                          KITTEN NUMBER                                                                              STRAIN   TROPHOZOITE COUNT                                       ______________________________________                                        1            WB       8.75 × 10.sup.3                                   2            WB       5.0 × 10.sup.3                                    3            S2       0                                                       4            S2       2.0 × 10.sup.4                                    5            D3       0                                                       6            D3       0                                                       ______________________________________                                    

9. Enzyme linked immunosorbent assay (ELISA)

Cat and dog gut mucosal homogenate samples were prepared by homogenizingtissue at 10% weight per volume in 2 mM EDTA, then stored at -80° C.Samples were thawed, diluted 1:2 (cat) or 1:1 (dog) with PEPBS (2 mMEDTA, 1 mM PMSF), the mixture dispersed by five passes through an 18 Gneedle, then spun at 17,000× g for 20 minutes. The supernatants, whichcontained soluble protein fractions, were used to perform the ELISA.Serum and bile samples were stored at -80° C., thawed, diluted 2-foldwith PEPBS, centrifuged, and supernatants removed for use in performingthe ELISA. All samples were assayed in duplicate.

Blocking for all assays other than dog IgG serum assays was done with 2%gamma globulin- free FCS in PBS for one hour at 37° C. with 3 washes.Dog IgG serum assays were blocked with 10% skim milk powder in PBS forone hour at 37° C.

For cat IgG assays, sonicated Giardia (S2) was used as the antigen.Sonicated S2 (0.2 mg/ml in PBS) was added to each well (100 μl per well)and incubated at 4° C. overnight. The wells were then washed (3 times)with PBS-tween. Serum, bile or gut mucosal supernatants prepared aspreviously described were used as the primary antibody (37° C., 1 hour,3 washes). Goat anti-cat IgG-HRP (KPL cat. #042002) diluted 1:1000 inPBS was used as the secondary antibody (37° C., 1 hour, 4 washes).

For IgA cat assays, sonicated Giardia S2 strain was used as the antigen.Serum bile or gut mucosal supernatants prepared as previously describedwere used as the primary antibody. Goat anti-cat IgA (Bethyl Lab. cat#A20-101) diluted 1:250 in PBS was used as the secondary antibody (37°C., 1 hour, 4 washes). Rabbit anti-goat IgA-HRP (Sigma cat. #A-4174)diluted 1:500 in PBS (37° C., 1 hour, 4 washes) was used as the tertiaryantibody.

For dog assays, sonicated Giardia S2 strain was used to coat the well asdescribed in the cat assay. Serum bile or gut mucosal supernatantsprepared as previously described were used as the primary antibody. Goatanti-dog IgG-HRP (KPL cat #041902) or goat antidog IgA-HRP(Bethyl Lab.Cat. #A40-104P) diluted 1:1000 in PBS was used as the secondaryantibody.

EXAMPLE 1 Strain Selection and Preservation

A variety of strains of Giardia lamblia were grown in vitro in tubes ofTYI-S-33 medium using our standard laboratory methodology. Primary andsecondary stocks were stored frozen at -70° C. Growth curves of thesethree strains of Giardia lamblia (WB, S2, D3) have been determined(FIG. 1) and compared to toxin response (cell elongation) of ChineseHamster Ovary (CHO) cells (FIGS. 2-4). Although D3 showed the mostimpressive growth characteristics, all three strains appeared promisingbased on adequate growth abilities in vitro, ability to infect targetanimal species, and toxic effects on CHO cells.

EXAMPLE 2 Kitten Vaccination--Experimental Infection

For this investigation, the following experimental groups wereestablished: Group A--7 kittens were sham-immunized with 0.2 ml of thepreviously described adjuvant and 0.8 ml PBS administeredsubcutaneously, then infected with Giardia lamblia strain S2 byintraduodenal inoculation of 1×10⁶ viable trophozoites in 1.0 ml PBS onday 28; Group B--3 kittens were vaccinated with 0.2 ml adjuvant and 0.8ml concentrated (10×) culture supernatant of S2 (750 μg protein/ml anddemonstrated CHO cell toxic activity) administered subcutaneously,boosted with the same preparation on day 21, then infected with S2intraduodenally on day 28; Group C--8 kittens were vaccinated with 0.2ml adjuvant and 0.8 ml sonicated whole S2 trophozoites (750 μgprotein/ml) administered subcutaneously, boosted with the samepreparation on day 21, then infected intraduodenally with S2 on day 28.

Clinical signs were monitored and quantitative fecal cyst counts wereperformed daily for 42 days following infection. The kittens wereweighed daily and growth curves generated for the non-vaccinated group(Group A) and the S2 sonicate vaccine group (Group C). Serum sampleswere obtained weekly and at post mortem for IgG ELISA titers. Aftereuthanasia, gut samples (duodenum, jejunum, ileum) were taken fortrophozoite counts, light microscopy, and electron microscopy. Mucosalscrapings, serum samples and bile were collected and stored frozen (-70°C.) for immunological analyses and enzymatic investigations.

Intermittent diarrhea or soft stools were seen irregularly in allkittens in this study. No differentiation of severity was observedbetween non-vaccinated animals and vaccinated animals. The weight gaindata for the non-vaccinated Group A and sonicated S2 vaccinated Group Bafter infection with Giardia lamblia are shown in FIG. 5. Vaccinatedkittens gained significantly more weight during the study thannon-vaccinated controls. Immunological responses in kittens of these 3experimental groups, as determined by serum IgG ELISA, are shown in FIG.6. Both Group B (toxin immunized) and Group C (sonicated S2-immunized)showed a significant increase in serum IgG, a typical response toimmunization and boosting. Only a minimal increase in titer was seen inthese immunized animals post-infection with Giardia. In contrast, theGroup A or non-immunized infected kittens showed a response only afterinfection, but this was a minimal increase in serum titer. Also includedin FIG. 6 are the IgG titers of a single (n=1) non-immunized andnon-infected kitten. Intestinal trophozoite counts were performed on 1.0cm gut segments from the duodenum, jejunum and ileum of all kittens. Theresults are illustrated in FIG. 7. Immunization with sonicated S2trophozoites (Group C) reduced the number of trophozoites present in thegut lumen of the duodenum and jejunum when compared to non-vaccinatedkittens (Group A). Data from toxin-immunized (Group B) animals is lessconvincing, possibly due to the comparatively smaller "n" value (n=3) ofthis group. Alternatively, immunization with toxin alone may preventsymptoms but not infection, or a higher dose of toxin may be moreeffective. Interestingly, trophozoites were not readily found in theileum as has been typically described for giardiasis in domestic cats³⁰.A variation of the intestinal trophozoite data described above is alsoshown in FIG. 8, where the percentage of gut samples in whichtrophozoites were seen are compared among experimental groups. Acomparatively small percentage of gut samples were positive for thepresence of Giardia trophozoites in the sonicated S2 immunized (Group C)kittens when compared to nonimmunized (Group A) and toxin-immunized(Group B) kittens.

Immunization with sonicated S2 trophozoites reduced the severity of cystexcretion in kittens.

Microscopic examination of select intestinal tissue samples indicatedthat trophozoites were present in non-immunized kittens but not presentor infrequently observed in sonicated S2-immunized kittens.

The results of immunological studies of IgA levels in serum and gutmucosa are presented in Table 4. No IgA response was noted inunvaccinated, unchallenged animals. A strong IgA response was noted inanimals vaccinated with S2 sonicate and challenged. In contrast,non-vaccinated challenged animals showed only a weak IgA response. Therewas a moderate IgA response in toxin vaccinated and challenged animals.Thus, vaccination induced a stronger IgA immune response than naturalinfection. A nonspecific immune response was noted in all bile samples,including in non-vaccinated, non-challenged animals.

                  TABLE 4                                                         ______________________________________                                        Serum and Mucosal Antibody IgA Titers                                                     Cat            Duoden                                             Group       No.    Serum   um     Jejunum                                                                              Ileum                                ______________________________________                                        Non-Vaccinated and                                                                        1      0       0      0      0                                    not challenged                                                                Non-Vaccinated and                                                                        2      80      80     10     20                                   challenged  3      40      40     20     40                                   Vaccinated with                                                                           4      800     40     80     20                                   toxin and   5      120     40     0      10                                   Challenged  6      600     40     80     80                                   Vaccinated with S2                                                                        7      4,000   160    160    80                                   Sonicate and                                                                              8      800     80     80     40                                   Challenged  9      1,200   80     80     20                                               10     4,000   320    40     320                                              11     1,000   160    160    160                                              12     2,000   640    160    160                                              13     2,000   80     160    320                                              14     3,200   320    160    640                                  ______________________________________                                    

EXAMPLE 3 Dog vaccination--Experimental Infection

A study of the efficiency of vaccination with sonicated Giardia strainS2 in dogs was performed as follows: Two groups of four puppies, 8 to 10weeks of age, were used. One group of animals (numbers 1 to 4) received,subcutaneously, a vaccine containing sonicated Giardia strain S2prepared as described previously (day 1). Each animal received 1 ml ofvaccine containing 600 μg protein in 0.8 ml PBS and 0.2 ml of thepreviously described adjuvant. The other group (numbers 5 to 8) was notvaccinated.

Three weeks later (day 21), animals 1 to 4 received a boostervaccination of the Giardia vaccine as above. Animals 5 to 8 were notvaccinated. On day 28, all pups were challenged with 4×10⁶ Giardialamblia strain N trophozoites. Strain N was used because it is highlyinfective in dogs. The challenge organisms were administeredintraduodenally in 1.0 ml PBS. Results show that immunization withGiardia strain S2 is cross protective against infection with Giardiastrain N.

Following challenge, blood samples were collected weekly, feces wereexamined daily for consistency, and cyst shedding until sacrifice of allanimals on day 56.

Vaccination protected dogs against diarrhea, with vaccinated animalshaving diarrhea for a mean of only 2.2±1.5 days while unvaccinatedanimals exhibited diarrhea for a mean of 8.2±5.6 days.

Cyst output data showed that vaccinated animals shed fewer cysts thanunvaccinated animals. Results are shown in FIG. 9.

Gut segments 1 cm in length were suspended in PBS and shaken for onehour at 37° C. The number of trophozoites were counted on ahemocytometer. Results are shown in Table 5. Vaccination eliminatedGiardia trophozoites from all areas of the intestine of immunizedanimals.

                  TABLE 5                                                         ______________________________________                                        Trophozoite Counts per cm Intestine                                           Immunized          Non-immunized                                              Duo-                       Duo-                                               denum     Jejunum  Ileum   denum Jejunum                                                                              Ileum                                 ______________________________________                                        Mean  0       0        0     0     30000  313                                 SD    0       0        0     0     44200  625                                 SE    0       0        0     0     22100  313                                 ______________________________________                                    

The vaccine induced a specific serum and mucosal immune response toGiardia. There was a strong serum IgG response to the vaccine startingthree weeks after vaccination. In contrast, unvaccinated dogs had a weakresponse only after four weeks of infection.

There was a weak to moderate serum IgA response after vaccination andchallenge in week six, but no serum IgA response in unvaccinatedanimals. Both groups had detectable IgA in the bile at post mortem. IgAwas detected in the duodenum and ileum in vaccinated and unvaccinatedanimals.

EXAMPLE 5 Production, Purification and Assay of Toxin

The WB isolate of Giardia lamblia was cultured at 37° C. for 10 days inTYI-S-33 media supplemented with NCTC-109 vitamin mix (3%), CLEX (10%),bile (0.8 g/l) and Piperacillin. Cultures were maintained in log phasewith transfers at 3 day intervals. After 10 days, the medium wascentrifuged at 3,000 × g for 15 minutes, sterile filtered (0.22 μ filter-Nalgene Co.) and concentrated with an Amicon apparatus with YM-10membrane filter.

The Chinese Hamster Ovary cell line CHO-K-1 (ATCC No. CCL61) was grownin Eagles alpha minimal essential medium (MEM) supplemented with 10%fetal calf serum (FCS) and 0.5% penicillin and streptomycin with aminoacids in 5% CO₂ at 37° C. with 90% relative humidity.

For morphological studies, a suspension of 5×10³ CHO cells in 200 μl ofmedia was added to each well of a 96 well tissue culture plate (LinbroCorp.) and incubated in 5% CO.sub. 2 at 37° C. for 48 hours. The mediumin each well was collected with a pipette and replaced with 150 μl offresh medium. Fifty μl of the substance to be tested was added to thefirst well of a row and a serial four-fold dilution was performed to theend of the row (12 wells). The plate was again incubated for 48 hours.Each well was then stained with Giemsa stain and the percentage ofelongated cells was determined. Each morphological datum on the graphrepresents the mean percentage of cells elongated from 500 cells counted(FIG. 10). Results of assay of Giardia lamblia concentrated medium werecompared to a control of concentrated Giardia-free medium. Cholera toxin(Sigma Corp.) and pertussis toxin were used as positive controls.Cholera toxin elicits a characteristic elongation reaction and pertussistoxin elicits a characteristic clumping reaction.

The effect of varying dilutions of the filtrates on the morphology ofCHO cells is illustrated in FIG. 10. The elongation of the cells isstriking after exposure to concentrated Giardia 10 day media (GM). CHOcells also displayed elongation, but not to the same degree, whenexposed to an identical dilution of concentrated control sterile medium(CM). The CM showed some morphological response at the higherconcentrations, but even the greatest response resulted in elongation ofonly 31.6% of CHO cells at a concentration of 0.25× original conc. TheGM had a much greater effect on CHO cell morphology at the higherconcentration (75% at 0.25× original conc.) and also produced elongationat very low concentrations. Elongation of CHO cells resulted atconcentrations above 1.5×10⁻⁵ × the original concentrated Giardia media.The elongation produced by the GM increased after the first dilution andthen began to decrease with subsequent dilutions (FIG. 10).

Five hundred thousand trophozoites were inoculated at day 0 into 16.0 mlof fresh TYI-S-33 medium in 10 culture tubes. Trophozoites were counteddaily from one tube randomly selected. A 2.0 ml sample of the medium waswithdrawn and sterile filtered with a 0.22 μ filter (Costar Corp.). Oneml of the sterilized culture medium was refrigerated at 4° C. and theother 1.0 ml was frozen at -70° C. This procedure was repeated for eightdays. Each fraction of culture medium collected was assayed as describedabove with the CHO cell bioassay.

Healthy log phase Giardia trophozoites were washed twice in 20 mM Trisbuffer, pH 6.0, centrifuged for 15 min. at 5,000× g, then resuspended in1.0 ml of buffer. The trophozoites were then sonicated by one 15 sec.burst and the sonicate examined by light microcopy for survivingtrophozoites. The sonicate was filtered through a 0.22 μ filter (CostarCorp.) to collect the cytosolic contents. The filter was backwashed with1.0 ml of buffer to collect the membranes and cytosolic fractions. Thebuffer, the cytosolic fraction, and the membrane fraction were assayedfor toxic activity with the CHO cell bioassay.

The growth media (GM) titer and Giardia growth curve relationship isdepicted in FIG. 11. The Giardia trophozoites divide and increase innumber in a logarithmic fashion. There is a lag phase evident on dayone, an acceleration phase on day two, a deceleration phase on daythree. Thereafter, the trophozoite number declines steadily. Thetrophozoites reached a maximum number of 4×10⁷ at day three. The titerof the media began to increase after day one and increase during thegrowth phase of the trophozoites. After day three, when the trophozoitenumber began to decline, the titer continued to increase.

Proteins in the toxin medium were separated using sodium dodecylsulphatepolyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained withCoomassie brilliant blue. The control medium was compared to Giardiasonicate (cytosolic contents), toxin medium that had Giardia lambliagrowing in it for 3, 4, 5, 6, 7, 10, and 25 days as well as FCS. Mediacontaining toxin contains proteins of approximate molecular weight of22, 32, 38, 39, and greater than 97 kDa that were not present in thecontrol media.

The Tris buffer and the membrane and cytoskeletal fractions from thesonicated trophozoites were negative for toxic activity. The cytosolicfraction resulted in cell death at the highest concentration andsubsequent cell survival and elongation at lower concentrations. Thesefindings were similar to the results obtained with GM.

The effect of bile on toxin production is shown in FIG. 12. Giardiagrown in media containing bile produced a toxin, as measured by CHO cellelongation. Giardia grown in media without bile did not.

EXAMPLE 6 Adult Cat Immunization

An additional study was conducted to investigate the immunologicalresponse of adult cats to immunization with extracts of the 3 strains ofGiardia lamblia. Twelve adult cats were immunized with sonicated Giardialamblia strains WB (373 μg protein/mL) (n=4), S2 (864 μg protein/mL)(n=4), and D3 (120 μg protein/mL) (n=4). Cats were vaccinatedsubcutaneously, receiving 1.0 ml of vaccine which comprised 0.2 ml ofthe previously described adjuvant and 0.8 ml sonicate. The cats wereboosted 21 days later and boosted a second time after an additional 21days. A pre-immunization blood sample was taken and all cats weresampled for IgG ELISA 7 days after each booster immunization. Thefollowing table (Table 6) is a summary of the immunological dataobtained from this study. Mature cats had a higher initial IgG titer toGiardia than the young kittens described in the above experiment (i.e.Table 2) and these adult cats responded to vaccination with asignificant increase in IgG titer.

                  TABLE 6                                                         ______________________________________                                        Serum IgG Titers from Adult Cats Immunized With Giardia                       CAT               PREIMMUNE   DAY 28  DAY 42                                  NUMBER  STRAIN    TITER       TITER   TITER                                   ______________________________________                                        9       WB        128         1280    nd                                      10      WB        128         1600    1600                                    13      WB        128         128     800                                     14      WB        64          3200    3200                                    6       S2        64          6400    3200                                    8       S2        400         3200    3200                                    12      S2        32          1280    6400                                    15      S2        32          1280    1600                                    3       D3        16          640     1600                                    5       D3        64          640     1600                                    11      D3        128         1280    800                                     18      D3        128         640     3200                                    ______________________________________                                         nd = not done                                                            

Modification of the above-described modes of carrying out the variousembodiments of this invention will be apparent to those skilled in theart following the teachings of this invention as set forth herein. Theexamples described above are not limiting, but are merely exemplary ofthis invention, the scope of which is defined by the following claims.

What is claimed is:
 1. A vaccine composition comprising a protectivelyimmunogenic amount of a giardia strain and an adjuvant, wherein saidgiardia strain is cultured in media containing bile so as to make itprotectively immunogenic and said giardia strain is disrupted and/orinactivated before use in said composition.
 2. The composition of claim1 which provides cross-protective immunity against giardia other thansaid giardia in said composition.
 3. The composition of claim 1 which isfor use in canine and feline species.
 4. The composition of claim 1wherein said giardia strain is selected from the group consisting of WB,S2 and D3.